Fixation and imaging of biological elements: heavy metals, diffusible substances, ions, peptides, and lipids

We tested various fixation and analysis methods to demonstrate by electron microscopy elemental imaging in tissues and cells, i.e., soluble substances such as many kinds of ionic elements, water soluble low molecular peptides, and even organic solvent soluble substances such as lipids. For the ionic elements, we tested frozen dried or freeze-substituted methods and organic or inorganic special chemical precipitation methods combined with microwaved fixation methods. The data were analyzed with electron beam X-ray microanalysis, electron energy filtered imaging analysis, and electron microscope autoradiography. The data were demonstrated as elemental distribution images and were calculated quantitatively. For the soluble low molecular peptides, we developed a tannic acid and aldehyde method combined with microwaved fixation. We discuss the theoretical background of the tannic acid fixation and microwaved fixation methods. For the organic solvent soluble substances, i.e., lipids including steroids, we successfully tested the use of a mixed fixative of aldehyde and osmium, digitonization, and osmification with the use of p-phenylendiamine or imidazole. We also proposed some new ideal biotracers for electron beam X-ray microanalysis and electron energy filtered imaging analysis.     

A Fine Structural Study and Energy-dispersive X-ray Spectrometrical Analysis of the Metals Present in Ovarian and Other Tissues of Normal and Lead-poisoned Quail (Coturnix coturnix japonica)

Abstract The fine structure was investigated of body tissues from Japanese quails which had received lead acetate added to their diet. A double fixation using glutaraldehyde and postfixation in osmic acid was compared with a fixation in glutaraldehyde alone. Because of the lack of contrast obtained in the specimens from the latter fixation, the presence of metal residues within the tissues were readily observable under the electron microscope. The various metal residues found in storage bodies in ovarian tissues from lead-poisoned quails were analyzed by means of energy-dispersive X-ray spectrometrical analysis. Out of a total of eight electron-dense, metal-containing inclusions which were analyzed, two contained lead. Other elements found in the inclusions were chlorine, phosphorus, iron and calcium, the latter element not being present in detectable amounts in those inclusions which also contained lead. Some correlation was found between the morphology and the elemental composition of the metal residues.     

X-ray microanalysis of elements in epon-embedded animals tissues]

X-ray microanalysis was performed on ultrathin sections of tissues from six rats, four of which having been exposed to experimental metabolic disorder by overdoses of NaCl and Ca-Vit. D2, respectively. It was demonstrated that the elements Cl, Si, P, S, Ca and traces of Fe are identifiable quantitatively in arteriolar walls of colon, heart and kidney even after conventional electron microscopic preparation (fixation, dehydration and Epon embedding). Depending on the pretreatment considerable differences in elemental quantities between animal groups were observed. The reliability of these findings must be confirmed by further tests on a greater number of individuals.     

Endogenous elements in the prostate. An X-ray microanalytical study of freeze-dried frozen sections and histochemical localization of zinc by potassium pyroantimonate

Summary Freeze-dried frozen sections prepared from unfixed rat lateral prostate were examined by X-ray microanalysis in an attempt to establish thein vivo distribution of endogenous ions. Poor morphological resolution was a limiting factor in the analysis of subcellular regions of the tissue. Glutaraldehyde fixation prior to cryo-sectioning resulted in considerable loss of elements. The results are discussed and compared with those obtained from ultrathin sections of tissue treated with potassium pyroantimonate. Using the latter method, it was possible to demonstrate a subcellular distribution pattern for the element zinc and to correlate the metal with specific organelles. It is considered that, unlike a number of other tissues, the rat prostate does not lend itself readily to cryoultramicrotomy as a preparative regime.     

X-ray microanalysis of biological specimens by high voltage electron microscopy

For the purpose of analyzing and imaging chemical components of cells and tissues at the electron microscopic level, 3 fundamental methods are available, chemical, physical and biological. Among the physical methods, two methods qualifying and quantifying the elements in the structural components are very often employed. The first method is radioautography which can demonstrate the localization of radiolabeled compounds which were incorporated into cells and tissues after the administration of radiolabeled compounds. The second method is X-ray microanalysis which can qualitatively analyze and quantify the total amounts of elements present in cells and tissues. We have developed the two methodologies in combination with intermediate high or high voltage transmission electron microscopy (200–400 kV) and applied them to various kinds of organic and inorganic compounds present in biological materials. As for the first method, radioautography, I had already contributed a chapter to PHC (37/2). To the contrary, this review deals with another method, X-ray microanalysis, using semi-thin sections and intermediate high voltage electron microscopy developed in our laboratory.

X-ray microanalysis is a useful method to qualify and quantify basic elements in biological specimens. We first quantified the end-products of histochemical reactions such as Ag in radioautographs, Ce in phosphatase reaction and Au in colloidal gold immunostaining using semithin sections and quantified the reaction products observing by intermediate high voltage transmission electron microscopy at accelerating voltages from 100 to 400 kV. The P/B ratios of all the end products Ag, Ce and Au increased with the increase of the accelerating voltages from 100 to 400 kV. Then we analyzed various trace elements such as Zn, Ca, S and Cl which originally existed in cytoplasmic matrix or cell organelles of various cells, or such elements as Al which was absorbed into cells and tissues after oral administration, using both conventional chemical fixation and cryo-fixation followed by cryo-sectioning and freeze-drying, or freeze-substitution and dry-sectioning, or freeze-drying and dry-sectioning producing semithin sections similarly to radioautography. As the results, some trace elements which originally existed in cytoplasmic matrix or cell organelles of various cells in different organs such as Zn, Ca, S and Cl, were effectively detected. Zn was demonstrated in Paneth cell granules of mouse intestines and its P/B ratios showed a peak at 300 kV. Ca was found in human ligaments and rat mast cells with a maximum of P/B ratios at 350 kV. S and Cl were detected in mouse colonic goblet cells with maxima of P/B ratios at 300 kV. On the other hand, some elements which were absorbed by experimental administration into various cells and tissues in various organs, such as Al in lysosomes of hepatocytes and uriniferous tubule cells in mice was detected with a maximum of P/B ratios at 300 kV.

From the results, it was shown that X-ray microanalysis using semi-thin sections observed by intermediate high voltage transmission electron microscopy at 300–400 kV was very useful resulting in high P/B ratios for quantifying some trace elements in biological specimens. These methodologies should be utilized in microanalysis of various compounds and elements in various cells and tissues in various organs.     

Phase-Contrast Hounsfield Units of Fixated and Non-Fixated Soft-Tissue Samples

X-ray phase-contrast imaging is a novel technology that achieves high soft-tissue contrast. Although its clinical impact is still under investigation, the technique may potentially improve clinical diagnostics. In conventional attenuation-based X-ray computed tomography, radiological diagnostics are quantified by Hounsfield units. Corresponding Hounsfield units for phase-contrast imaging have been recently introduced, enabling a setup-independent comparison and standardized interpretation of imaging results. Thus far, the experimental values of few tissue types have been reported; these values have been determined from fixated tissue samples. This study presents phase-contrast Hounsfield units for various types of non-fixated human soft tissues. A large variety of tissue specimens ranging from adipose, muscle and connective tissues to liver, kidney and pancreas tissues were imaged by a grating interferometer with a rotating-anode X-ray tube and a photon-counting detector. Furthermore, we investigated the effects of formalin fixation on the quantitative phase-contrast imaging results.     

Determination of metallic ion transfer from an implanted prosthesis by the PIXE method

Prostheses can release some metallic elements to the surrounding tissues, particularly when they are not covered with a biomaterial layer and when an unsealing process happens. We try to measure major and trace elements in these tissues with an experimentally sensitive method. Proton-induced X-ray emission is used to detect about 10 elements in tissue. Tissues are calcinated and deposited in a thin layer before irradiation. Results are obtained in a standard and samples from three patients. We observe contamination by Ti, Cr, Ni, and Zn in the tissues. Correlations are to be studied between these atomic transfers and prosthesis in the patient.     

The effect of fixative tonicity on the myosin filament lattice volume of frog muscle fixed following exposure to normal or hypertonic Ringer

Synopsis Frog sartorius muscles have been fixed sequentially with acrolein and osmium tetroxide dissolved in vehicles of various tonicities, and the myosin filament spacings and sarcomere lengths measured with the electron microscope. From these dimensions the myosin unit-cell volume has been calculated and compared with X-ray diffraction data to determine the effect of fixation. In muscles soaked in normal Ringer and afterwards fixed using normal Ringer as a vehicle for the fixation agents, the unitcell volume undergoes a 10.4% reduction during the preparative procedure. Muscles soaked in hypertonic Ringer undergo a similar reduction in volume during fixation, provided hypertonic Ringer is used as the vehicle; if they are fixed in normal Ringer, the lattice swells during fixation, even if the change to the normal tonicity vehicle occurs after acrolein fixation. If blocks suitable for embedding are cut from the muscles before, rather than after, osmium fixation, more complex changes in intracellular dimensions may occur, including artefactual swelling of the T-system. It is concluded that fixation of tissues exposed to modifications of normal physiological solutions should be performed using the same modified solutions as fixative vehicles.     

Effect of inoculation on the elemental composition ofMedicago sativa

Inoculation with rhizobia ofM. sativa plants grown either in the laboratory on nitrogen-free agar and soil-agar slopes or in field experiments, decreases in all cases the concentration of Mo in plant shoots and increases that of Rb, while differences in the concentration of several elements with atomic numbers between 17 and 42 could also be identified by X-ray fluorescence. These elemental alterations depend on the fixation induced either by inoculation or by the effective indigenous rhizobial population of each growth medium and are not caused by rhizosphere pH alterations but seem to be directly related to the induced biological nitrogen fixation.     

X-ray fluorescence-based differentiation of neck tissues in a bovine model: Implications for potential intraoperative use

This study explores the possibility of using X-ray fluorescence (XRF)-based trace-element analysis for differentiation of various bovine neck tissues. It is motivated by the requirement for an intra-operative in-vivo method for identifying parathyroid glands, particularly beneficial in surgery in the central neck-compartment. Using a dedicated X-ray spectral analysis, we examined ex-vivo XRF spectra from various histologically verified fresh neck tissues from cow, which was chosen as the animal model; these tissues included fat, muscle, thyroid, parathyroid, lymph nodes, thymus and salivary gland. The data for six trace elements K, Fe, Zn, Br, Rb and I, provided the basis for tissue identification by using multi-parameter analysis of the recorded XRF spectra. It is shown that the combination of XRF signals from these elements is sufficient for a reliable tissue differentiation. The average total abundance of these trace elements was evaluated in each tissue type, including parathyroid and salivary gland for the first time. It is shown that some tissues can unequivocally be identified on the basis of the abundance of a single element, for example, iodine and zinc for the identification of thyroid gland and muscle, respectively.     

X-ray microanalysis of zinc and calcium in ultrathin sections of human sperm cells using the pyroantimonate technique.

X-ray microanalysis of pyroantimonate-fixed sperm cells indicates the retention of calcium and zinc subcellularly in similar proportions to air dried cells. The ultrastructure is well preserved and is corelated with the analysis. Sodium, potassium and chlorine are all removed during the fixation. CAlcium and zinc are found present intracellularly both in association with and independent of antimonate precipitation. There thus appears to be a varying degree of binding of those elements subcellularly, precipitation occurring where binding is reduced.     

A new technique for visualizing proanthocyanidins by light microscopy

We describe a new technique for visualizing proanthocyanidin-containing elements in plant tissues. Our innovation is the fixation of condensed tannins with an exogenous protein prior to alcohol dehydration. In this way, tannins do not undergo partial solubilization during the dehydration sequence and appear as sharply contoured globules of various diameters.     

Sample preparation of animal tissues and cell cultures for secondary ion mass spectrometry (SIMS) microscopy.

Sample preparation is a critical step in the elemental analysis of animal tissues and cell cultures with ion microscopy. Since live cells cannot be analyzed with ion microscopy, a careful sample fixation is necessary which preserves the native structural and chemical integrity of a specimen. The evaluation of morphological and chemical integrity of a fixed specimen is necessary before any physiological explanation of ion fluxes is interpreted based on ion microscopy. For diffusible ion localization studies, strict cryogenic procedures are recommended. Examples are shown for diffusible ion microanalysis in frozen-freeze-dried tissues and cell cultures. Ion microscopy studies of tightly bound elements/molecules may be conducted in chemically fixed and/or plastic embedded specimens. Since it is not generally known which elements/molecules are tightly bound to the tissue matrix, a confirmation of elemental distribution with cryogenic procedures is desirable. A recent approach of combining laser scanning confocal fluorescence microscopy and ion microscopy on the same frozen freeze-dried cell is also discussed for recognizing smaller cytoplasmic structures in ion microscopy images.     

Studies on Bovine Mast Cells: I. The Effect of Formalin Fixation

Studies are reported on the effect of aqueous formalin fixation on the bovine mast cell. Paraffin sections of bovine skin were prepared from tissues fixed for 7 to 72 hr in 10% neutral formalin in saline, and from tissues which were treated with water for various times both before and after formalin fixation. Metachromatic halos, scattering of the granules, and fraying of the cell outline, were seen in the mast cells of tissues washed in water before fixation. Exposure to water after fixation did not produce these artifacts. The tendency towards orthochromatic staining, and the occurrence of perinuclear clear zones are probably effects of the formalin and not of overstaining or of exposure of the tissue to water. The majority of the metachromatic material in the bovine mast cell is water soluble, and may be removed by washing in water before fixation, whereas the granules are relatively resistant to water. The optimum time of formalin fixation of bovine skin to permit studies on the metachromatic material of the mast cells was 24 to 36 hr.     

Towards a molecular description of intermediate filament structure and assembly

Intermediate filaments (IFs) represent one of the prominent cytoskeletal elements of metazoan cells. Their constituent proteins are coded by a multigene family, whose members are expressed in complex patterns that are controlled by developmental programs of differentiation. Hence, IF proteins found in epidermis differ significantly from those in muscle or neuronal tissues. Due to their fibrous nature, which stems from a fairly conserved central alpha-helical coiled-coil rod domain, IF proteins have long resisted crystallization and thus determination of their atomic structure. Since they represent the primary structural elements that determine the shape of the nucleus and the cell more generally, a major challenge is to arrive at a more rational understanding of how their nanomechanical properties effect the stability and plasticity of cells and tissues. Here, we review recent structural results of the coiled-coil dimer, assembly intermediates and growing filaments that have been obtained by a hybrid methods approach involving a rigorous combination of X-ray crystallography, small angle X-ray scattering, cryo-electron tomography, computational analysis and molecular modeling.     

Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos

Chromatin immunoprecipitation (ChIP) is widely used for mapping DNA-protein interactions across eukaryotic genomes in cells, tissues or even whole organisms. Critical to this procedure is the efficient cross-linking of chromatin-associated proteins to DNA sequences that are in close proximity. Since the mid-nineties formaldehyde fixation has been the method of choice. However, some protein-DNA complexes cannot be successfully captured for ChIP using formaldehyde. One such formaldehyde refractory complex is the developmentally regulated insulator factor, Elba. Here we describe a new embryo fixation procedure using the bi-functional cross-linking reagents DSG (disuccinimidyl glutarate) and DSP (dithiobis[succinimidyl propionate). We show that unlike standard formaldehyde fixation protocols, it is possible to capture Elba association with insulator elements in 2–5 h embryos using this new cross-linking procedure. We show that this new cross-linking procedure can also be applied to localize nuclear proteins that are amenable to ChIP using standard formaldehyde cross-linking protocols, and that in the cases tested the enrichment was generally superior to that achieved using formaldehyde cross-linking.     

Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos

Chromatin immunoprecipitation (ChIP) is widely used for mapping DNA-protein interactions across eukaryotic genomes in cells, tissues or even whole organisms. Critical to this procedure is the efficient cross-linking of chromatin-associated proteins to DNA sequences that are in close proximity. Since the mid-nineties formaldehyde fixation has been the method of choice. However, some protein-DNA complexes cannot be successfully captured for ChIP using formaldehyde. One such formaldehyde refractory complex is the developmentally regulated insulator factor, Elba. Here we describe a new embryo fixation procedure using the bi-functional cross-linking reagents DSG (disuccinimidyl glutarate) and DSP (dithiobis[succinimidyl propionate). We show that unlike standard formaldehyde fixation protocols, it is possible to capture Elba association with insulator elements in 2–5 h embryos using this new cross-linking procedure. We show that this new cross-linking procedure can also be applied to localize nuclear proteins that are amenable to ChIP using standard formaldehyde cross-linking protocols, and that in the cases tested the enrichment was generally superior to that achieved using formaldehyde cross-linking.     

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

The emergence of electron tomography as a tool for three dimensional structure determination of cells and tissues has brought its own challenges for the preparation of thick sections. High pressure freezing in combination with freeze substitution provides the best method for obtaining the largest volume of well-preserved tissue. However, for deeply embedded, heterogeneous, labile tissues needing careful dissection, such as brain, the damage due to anoxia and excision before cryofixation is significant. We previously demonstrated that chemical fixation prior to high pressure freezing preserves fragile tissues and produces superior tomographic reconstructions compared to equivalent tissue preserved by chemical fixation alone. Here, we provide further characterization of the technique, comparing the ultrastructure of Flock House Virus infected DL1 insect cells that were (1) high pressure frozen without fixation, (2) high pressure frozen following fixation, and (3) conventionally prepared with aldehyde fixatives. Aldehyde fixation prior to freezing produces ultrastructural preservation superior to that obtained through chemical fixation alone that is close to that obtained when cells are fast frozen without fixation. We demonstrate using a variety of nervous system tissues, including neurons that were injected with a fluorescent dye and then photooxidized, that this technique provides excellent preservation compared to chemical fixation alone and can be extended to selectively stained material where cryofixation is impractical.     

Energy-dispersive x-Ray Analysis of Phosphorus, Potassium, Magnesium, and Calcium in Globoid Crystals in Protein Bodies from Different Regions of Cucurbita maxima Embryos

The seeds of Cucurbita maxima contain protein bodies with electrondense globoid crystals. Because of their density globoid crystals are ideal material for energy-dispersive x-ray (EDX) analysis studies of elemental composition. Fixation trials were carried out to test globoid crystal extraction during glutaraldehyde fixation, water washing, and ethanol dehydration. Glutaraldehyde fixation without subsequent washing or dehydration alone produced no significant changes in elemental composition of cotyledon globoid crystals. If glutaraldehyde fixation was followed by water washes or ethanol dehydration there was some loss of the major globoid crystal elements but the relative percentages of the elements P, K, Ca, and Mg remained relatively unchanged. In this paper results of a study of the P, K, Mg, and Ca content of globoid crystals in different tissues of squash embryos are presented. The globoid crystals in the radicle were found to be the least dense in the embryo. Globoid crystals from all embryo regions contained P, K, and Mg. In the various embryo regions P and Mg maintained relatively constant proportions of the globoid crystal composition while K and Ca varied. Of particular significance is the distribution of Ca which is generally an immobile element. Calcium was found in highest amounts in the globoid crystals of the radicle and stem regions while globoid crystals in much of the cotyledon contained little, if any, Ca. The Ca storage thus seems to be spatially arranged in a manner that would aid early growth of the root-shoot axis.